Endogenous Insulin-like Growth Factor Binding Protein-3 Research Articles

Insulin-like growth factor binding protein-3 links obesity and breast cancer progression.

Obesity is associated epidemiologically with poor breast cancer prognosis, but the mechanisms remain unclear. Since IGF binding protein-3 (IGFBP-3) influences both breast cancer growth and adipocyte maturation, it may impact on how obesity promotes breast oncogenesis. This study investigated the role of endogenous IGFBP-3 on the development of obesity and subsequently on breast tumor growth. Wild-type (WT) C57BL/6 or IGFBP-3-null (BP3KO) mice were fed a high-fat diet (HFD) or control chow-diet for 15 weeks before orthotopic injection with syngeneic EO771 murine breast cancer cells. When the largest tumor reached 1000 mm3, tissues and tumors were excised for analysis. Compared to WT, BP3KO mice showed significantly reduced weight gain and mammary fat pad mass (contralateral to tumor) in response to HFD, despite similar food intake. EO771 tumor weight and volume were increased by HFD and decreased by BP3KO. Despite differences in tumor size, tumors in BP3KO mice showed no differences from WT in the number of mitotically active (Ki67+) and apoptotic (cleaved caspase-3+) cells, but had greater infiltration of CD3+ T-cells. These data suggest that endogenous (circulating and/or stromal) IGFBP-3 is stimulatory to adipose tissue expansion and enhances mammary tumor growth in immune-competent mice, potentially by suppressing T-cell infiltration into tumors.

Abstract 741: Insulin-like growth factor binding protein-3 (IGFBP-3) enhances obesity-related breast tumorigenesis

Abstract Introduction and Aim: Epidemiological studies have shown an association between obesity and poor breast cancer prognosis. We have reported that IGFBP-3 influences both breast cancer growth and adipocyte maturation. This study investigated the role of endogenous IGFBP-3 on the development of obesity and subsequently on breast tumor growth. Methods: Wild-type (WT) C57BL/6 or IGFBP-3 knock-out (BP3KO) mice were fed either a high-fat diet (HFD) or control chow diet for 15 weeks, then injected with the syngeneic mouse breast cancer cell line E0771 in the 4th left mammary gland. When the largest tumor reached 1000 mm3, tissues including the mammary fat pad were excised, formalin-fixed and paraffin-embedded for immunohistochemical analysis. Results and Conclusion: Compared to WT mice, BP3KO mice showed both significantly reduced weight gain and mammary fat pad mass (contralateral to tumor) in response to HFD (p < 0.0001, 1-way ANOVA), despite similar food intake between WT and BP3KO mice on HFD. E0771 tumor weight and volume were overall increased by HFD independent of IGFBP-3 status (p = 0.051), and decreased in BP3KO mice independently of diet (p < 0.001, 2-way ANOVA). Despite differences in tumor volumes and weights, tumors from WT and BP3KO mice did not show differences in either the number of mitotically active (Ki67+) or apoptotic (cleaved caspase-3+) cells. Tumors from BP3KO mice however, showed greater infiltration of CD3+ T-cells compared to WT mice (p = 0.0299, 2-way ANOVA). WT and BP3KO mice showed similar intra-tumoral mean vessel densities (CD31+ area) but differed in terms of the relationship between vessel density and final tumor volume. Increased vessel density was associated with decreased tumor volume in BP3KO mice (r = -0.62, p = 0.03, Spearman's correlation test) in contrast to the increased vessel density associated with increased tumor volume in wild-type mice (r = 0.63, p = 0.02, Spearman's correlation test). These data suggest that endogenous (circulating and/or stromal) IGFBP-3 (i) has a potentially stimulatory role in the expansion of adipose tissue, (ii) can enhance mammary tumor growth in immune-competent mice, potentially by influencing T-cell infiltration into the tumor and (iii) may influence the relationship between tumor vascularity and growth. Citation Format: Tiffany Scully, Carolyn D. Scott, Hasanthi C. de Silva, Sue M. Firth, Stephen M. Twigg, John E. Pintar, Robert C. Baxter. Insulin-like growth factor binding protein-3 (IGFBP-3) enhances obesity-related breast tumorigenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 741.

Maternally Administered Cyclic Glycine-Proline Increases Insulin-Like Growth Factor-1 Bioavailability and Novelty Recognition in Developing Offspring.

Cyclic glycine-proline (cGP), a metabolite of IGF-1, is an endogenous neuropeptide that improves memory in adult rats. The presence and concentrations of endogenous cGP, and its association with IGF-1 and IGF binding protein-3 (IGFBP-3) in rat milk and plasma, were evaluated during postnatal development. Maternal-infantile transfer of cGP during lactation and its efficacy on the memory of developing offspring were also investigated. Dams were gavaged with either cGP (3 mg/kg) or saline daily from postnatal days 8-22. Concentrations of cGP were measured in dams' milk, and concentrations of cGP, IGF-1, and IGFBP-3 were measured in the plasma of dams, pups, and young adults. The recognition memory, locomotor function, and anxiety-like behavior of offspring were evaluated using behavioral tests. Endogenous cGP was detected in rat milk, and its concentration was higher during peak lactation compared with late lactation. Comparisons within control groups showed low endogenous IGF-1 and IGFBP-3 and high endogenous cGP concentrations in the plasma of male pups. The reduced IGFBP-3 and increased cGP may be a response to increase the bioavailability of IGF-1 during infancy. Exogenous cGP showed oral bioavailability and effective maternal-infantile transfer through milk. Maternally transferred cGP also led to improved recognition memory in the developing offspring, possibly through increased IGF-1 bioavailability, with no effect on locomotor activity and anxiety-like behavior. These results show that cGP is an essential endogenous peptide during early postnatal development as it improves the bioavailability of IGF-1 during infancy. Furthermore, maternal cGP supplementation offers an effective and natural route of administration for improving memory in the developing offspring.

Involvement of p53 in insulin-like growth factor binding protein-3 regulation in the breast cancer cell response to DNA damage.

Chemotherapy drugs that induce apoptosis by causing DNA double-strand breaks, upregulate the tumor suppressor p53. This study investigated the regulation of the growth-regulatory protein insulin-like growth factor binding protein-3 (IGFBP-3), a p53 target, by DNA-damaging agents in breast cancer cells. IGFBP-3 was upregulated 1.4- to 13-fold in response to doxorubicin and etoposide in MCF-10A, Hs578T, MCF-7 and T47D cells, which express low to moderate basal levels of IGFBP-3. In contrast, IGFBP-3 was strongly downregulated by these agents in cells with high basal levels of IGFBP-3 (MDA-MB-231, MDA-MB-436 and MDA-MB-468). In MDA-MB-468 cells containing the R273H p53 mutation, reported to display gain-of-function properties, chemotherapy-induced suppression of IGFBP-3 was not reversed by the p53 reactivating drug, PRIMA-1, or by p53 silencing, suggesting that the decrease in IGFBP-3 following DNA damage is not a mutant p53 gain-of-function response. SiRNA-mediated downregulation of endogenous IGFBP-3 modestly attenuated doxorubicin-induced apoptosis in MDA-MB-468 and Hs578T cells. IGFBP-3 downregulation in some breast cancer cell lines in response to DNA-damaging chemotherapy may have clinical implications because suppression of IGFBP-3 may modulate the apoptotic response. These observations provide further evidence that endogenous IGFBP-3 plays a role in breast cancer cell responsiveness to DNA damaging therapy.

Inhibition of Insulin-like Growth Factor–Binding Protein-3 Signaling through Sphingosine Kinase-1 Sensitizes Triple-Negative Breast Cancer Cells to EGF Receptor Blockade

The type I EGF receptor (EGFR or ErbB1) and insulin-like growth factor-binding protein-3 (IGFBP-3) are highly expressed in triple-negative breast cancer (TNBC), a particularly aggressive disease that cannot be treated with conventional therapies targeting the estrogen or progesterone receptors (ER and PR), or HER2. We have shown previously in normal breast epithelial cells that IGFBP-3 potentiates growth-stimulatory signaling transduced by EGFR, and this is mediated by the sphingosine kinase-1 (SphK1)/sphingosine 1-phosphate (S1P) system. In this study, we investigated whether cotargeting the EGFR and SphK1/S1P pathways in TNBC cells results in greater growth inhibition compared with blocking either alone, and might therefore have novel therapeutic potential in TNBC. In four TNBC cell lines, exogenous IGFBP-3 enhanced ligand-stimulated EGFR activation, associated with increased SphK1 localization to the plasma membrane. The effect of exogenous IGFBP-3 on EGFR activation was blocked by pharmacologic inhibition or siRNA-mediated silencing of SphK1, and silencing of endogenous IGFBP-3 also suppressed EGF-stimulated EGFR activation. Real-time analysis of cell proliferation revealed a combined effect of EGFR inhibition by gefitinib and SphK1 inhibition using SKi-II. Growth of MDA-MB-468 xenograft tumors in mice was significantly inhibited by SKi-II and gefitinib when used in combination, but not as single agents. We conclude that IGFBP-3 promotes growth of TNBC cells by increasing EGFR signaling, that this is mediated by SphK1, and that combined inhibition of EGFR and SphK1 has potential as an anticancer therapy in TNBC in which EGFR and IGFBP-3 expression is high.

Interaction Between IGF Binding Protein-3 and TGFβ in the Regulation of Adipocyte Differentiation

The development of white adipose tissue involves both the hypertrophy of existing adipocytes and the proliferation and differentiation of preadipocytes. Adipogenic differentiation is inhibited by TGFβ signaling through Smad2/3, and IGF binding protein-3 (IGFBP-3) is also known to activate Smad2/3 signaling in some cell types. We previously reported that exogenous or overexpressed IGFBP-3 inhibits adipogenesis in 3T3-L1 cells, but the role of endogenous IGFBP-3 in this process, and its possible interaction with TGFβ, is not known. During 10-d adipogenic differentiation initiated by insulin, dexamethasone, and 3-isobutyl-1-methylxanthine, 3T3-L1 cells expressed increasing levels of IGFBP-3 and TGFβ1, secreting over 1000 pg/ml of both proteins. Exogenous recombinant human IGFBP-3 paralleled TGFβ1 in stimulating Smad2 phosphorylation in 3T3-L1 preadipocytes, but no additive effect was observed for the two agents. In contrast, knockdown of endogenous IGFBP-3 by small interfering RNA (siRNA) significantly impaired Smad2 activation by 0.25 ng/ml TGFβ1. Transient expression of human IGFBP-3 significantly inhibited the induction of adipogenic markers adiponectin and resistin, and the appearance of lipid droplets, but down-regulation of endogenous IGFBP-3 by siRNA had little effect on the expression of either marker during the 10-d differentiation, compared with nonsilencing control siRNA. However, down-regulation of endogenous IGFBP-3 using two different siRNA significantly reversed the inhibitory effect of TGFβ1 on both adiponectin and resistin induction. We conclude that IGFBP-3 activates inhibitory Smad signaling in 3T3-L1 cells and that endogenous IGFBP-3 modulates their adipogenic differentiation by regulating cell sensitivity towards the inhibitory effect of TGFβ.

Endogenous IGFBP-3 regulates excess collagen expression in intestinal smooth muscle cells of Crohnʼs disease strictures

Stricture formation occurs in ≈30% of patients with Crohn's disease (CD) and is a significant cause of morbidity. Strictures are characterized by intestinal smooth muscle cell hyperplasia, smooth muscle cell hypertrophy, and fibrosis due to excess net extracellular matrix production, including collagen. Transforming growth factor-β1 (TGF-β1) has profibrotic effects in many tissues due to its ability to regulate collagen expression and extracellular matrix dynamics. We previously showed that both insulin-like growth factor (IGF) binding protein-3 (IGFBP-3) and TGF-β1 are expressed by normal human intestinal smooth muscle cells, bind to, and activate TGF-βRII/I receptors in these cells. Smooth muscle cells isolated from the muscularis propria of patients were used to prepare RNA, protein lysates, or placed into primary culture. IGFBP-3, TGF-β1, and collagen IαI expression was measured with quantitative reverse-transcription polymerase chain reaction (RT-PCR) and protein levels by enzyme-linked immunosorbent assay (ELISA) or immunoblot. Expression and production of IGFBP-3, TGF-β1, and collagen IαI were significantly increased specifically in smooth muscle cells isolated from regions of strictured intestine in CD compared to nonstrictured histologically normal resection margin. IGFBP-3 and TGF-β1 regulated collagen IαI expression and production via a TGF-βRII/I-dependent and Smad2/3-dependent mechanism. Upregulated (excess) collagen IαI expression and production in smooth muscle cells of strictures and basal collagen IαI in smooth muscle cells of normal margin were inhibited by immunoneutralization of IGFBP-3 or TGF-β1. The findings indicate that upregulated endogenous IGFBP-3 and TGF-β1 expression regulates excess collagen IαI production and contributes to fibrosis and stricture formation in CD.

Biological effects induced by insulin‐like growth factor binding protein 3 (IGFBP‐3) in malignant melanoma

The insulin like growth factor (IGF) signaling pathway has been shown to contribute to melanoma progression, but little is known about the role of the IGF binding protein 3 (IGFBP-3) in melanoma biology. The aim of the present study was to characterize expression, function and regulation of IGFBP-3 in malignant melanomas and study its potential as a biomarker. The expression of IGFBP-3 varied between different human melanoma cell lines and reintroduction of the protein in non-expressing cells led to induction of apoptosis. Interestingly, in cell lines expressing endogenous IGFBP-3, siRNA silencing of the protein led to a cell line-dependent decrease in proliferation, but had no effect on apoptosis and invasion. Examination of patient material showed that IGFBP-3 is unexpressed in benign nevi while a slight increase in protein expression was seen in primary and metastatic melanoma. However, expression of the protein was low and no correlation was found with circulating levels of IGFBP-3 in serum, suggesting that IGFBP-3 has limited potential as a predictive marker in malignant melanoma. We showed that promoter methylation of IGFBP-3 occurred in both melanoma cell lines and patient material, implicating epigenetic silencing as a regulation mechanism. Furthermore, expression of the protein was shown to be regulated by the PI3-kinase/AKT and MAPK/ERK1/2 pathways. In summary, our findings suggest that IGFBP-3 can exert dual functional effects influencing both apoptosis and proliferation. Development of resistance to the antiproliferative effects of IGFBP-3 may be an important step in progression of malignant melanomas.

Effects of endogenous insulin-like growth factor binding protein-3 on cell cycle regulation in breast cancer cells

High tissue insulin-like growth factor binding protein-3 (IGFBP-3) expression in breast cancers is associated in some studies with rapid growth and poor outcome. This study examined the effects of endogenous IGFBP-3 in Hs578T breast cancer cells, which are IGF-unresponsive and grow aggressively despite relatively high IGFBP-3 expression. IGFBP-3 downregulation using siRNA was associated with increases in DNA synthesis, the percentage of cells in S phase and viable cell numbers, accompanied by increases in cyclins A and D1, a decrease in p27 expression, and increased phosphorylation of retinoblastoma (Rb) on Ser795. Downregulation of IGFBP-3 inhibited extracellular signal-regulated kinase (ERK) activation and cell migration in a monolayer wound healing assay. These results indicate that endogenous IGFBP-3 is anti-proliferative and pro-migratory in Hs578T cells and that these effects are IGF-independent. Poor outcome in breast tumours expressing high levels of IGFBP-3 may be due to the effects of IGFBP-3 on cell migration rather than cell growth.

Insulin-like growth factor binding protein-3 has dual effects on gastrointestinal stromal tumor cell viability and sensitivity to the anti-tumor effects of imatinib mesylate in vitro

BackgroundImatinib mesylate has significantly improved survival and quality of life of patients with gastrointestinal stromal tumors (GISTs). However, the molecular mechanism through which imatinib exerts its anti-tumor effects is not clear. Previously, we found up-regulation of insulin-like growth factor binding protein-3 (IGFBP3) expression in imatinib-responsive GIST cells and tumor samples. Because IGFBP3 regulates cell proliferation and survival and mediates the anti-tumor effects of a number of anti-cancer agents through both IGF-dependent and IGF-independent mechanisms, we hypothesized that IGFBP3 mediates GIST cell response to imatinib. To test this hypothesis, we manipulated IGFBP3 levels in two imatinib-responsive GIST cell lines and observed cell viability after drug treatment.ResultsIn the GIST882 cell line, imatinib treatment induced endogenous IGFBP3 expression, and IGFBP3 down-modulation by neutralization or RNA interference resulted in partial resistance to imatinib. In contrast, IGFBP3 overexpression in GIST-T1, which had no detectable endogenous IGFBP3 expression after imatinib, had no effect on imatinib-induced loss of viability. Furthermore, both the loss of IGFBP3 in GIST882 cells and the overexpression of IGFBP3 in GIST-T1 cells was cytotoxic, demonstrating that IGFBP3 has opposing effects on GIST cell viability.ConclusionThis data demonstrates that IGFBP3 has dual, opposing roles in modulating GIST cell viability and response to imatinib in vitro. These preliminary findings suggest that there may be some clinical benefits to IGFBP3 therapy in GIST patients, but further studies are needed to better characterize the functions of IGFBP3 in GIST.

Silibinin inhibits renal cell carcinoma via mechanisms that are independent of insulin‐like growth factor‐binding protein 3

To investigate whether silibinin, a flavonoid antioxidant with anticancer properties that inhibits cellular growth via the up-regulation of insulin-like growth factor binding protein 3 (IGFBP-3), inhibits renal cell carcinoma (RCC) growth via the IGFBP-3 pathway. Cell morphology, DNA synthesis by thymidine incorporation, viability assessed by 3,4,5 dimethylthiazol-2,5 diphenyl tetrazolium bromide (MTT) assays, trypan blue exclusion, and lactate dehydrogenase (LDH) release, apoptosis and IGFBP-3 protein (Western blotting) were evaluated in silibinin-treated SN12K1 cells (a cell line derived from metastatic RCC) in the presence or absence of IGFBP-3 immunoneutralization. Silibinin suppressed SN12K1 DNA synthesis at 24 and 48 h incubation by a mean (SD) of at least 68 (10)% of the control values at > or =2 micromol/L (P < 0.05). At high concentrations (>80 micromol/L) cell viability was compromised, as shown by decreased MTT uptake of 62 (10)% of control values (P < 0.001), reduced cell counts of > or =77 (9)% of control values (P < 0.05), elevated LDH release of 212 (49)% of control (P < 0.001), and the presence of necrotic and apoptotic cells on immunofluorescence staining. Removing endogenous IGFBP-3 by neutralizing with anti-IGFBP-3 antibody increased DNA synthesis by 240 (35)% of control for 24 h, (P < 0.001). However, on Western blot analysis of IGFBP-3 levels in conditioned media incubated in the presence of silibinin there was down-regulation of protein expression. Silibinin suppresses SN12K1 cell growth and, at high doses, increases cell death. These effects are independent of IGFBP-3.

IGF-I and IGFBP-3 augment transforming growth factor-β actions in human renal carcinoma cells

Renal cell carcinoma (RCC) is the most prevalent cancer of the kidney. In human RCC cells, we recently showed that insulin-like growth factor I (IGF-I) has growth-promoting effects regulated by IGF-binding protein 3 (IGFBP-3). In this study, the analysis was expanded to include the interaction between the IGF and transforming growth factor-beta (TGF-beta) systems in the human RCC cells Caki-2 (from a primary tumor) and SK-RC-52 (from a metastasis). Functional effects such as cell proliferation, TGF-beta receptor (TbetaR) signaling, and IGFBP-3 levels were monitored after stimulation with various concentrations of IGF-I, TGF-beta, and IGFBP-3. In addition, human RCC tissues as well as experimental human RCC tumors were analyzed for cellular expression of phosphorylated Smad2 by immunohistochemistry. TGF-beta regulated the endogenous IGFBP-3 levels in these RCC cells as neutralizing anti-TGF-beta(1-3) antibodies strongly reduced the basal IGFBP-3 level. In addition, IGF-I increased the IGFBP-3 levels five- to eightfold with TGF-beta acting in synergy to enhance the IGFBP-3 levels 12- to 17-fold. Neutralizing TGF-beta(1-3) activity circumvented the growth inhibitory effects of IGFBP-3 seen in SK-RC-52, whereas it inhibited the growth-promoting effects of IGFBP-3 in Caki-2. Moreover, IGF-I interacted directly with TGF-beta activation of the TbetaR complex by enhancing phosphorylation and nuclear translocation of Smad2. This study demonstrates a direct interaction of the IGF and TGF-beta systems in human renal carcinoma cells. The observations that IGF-I enhances the TGF-beta signaling and that TGF-beta promotes IGFBP-3 production and thus influence the biological activity of IGF may be of importance for future therapeutic options.

Growth Inhibitory Concentrations of Androgens Up-Regulate Insulin-Like Growth Factor Binding Protein-3 Expression via an Androgen Response Element in LNCaP Human Prostate Cancer Cells

IGF binding protein-3 (IGFBP-3), the most abundant circulating IGF binding protein, inhibits cell growth and induces apoptosis by both IGF-I-dependent and -independent pathways. The ability of IGFBP-3 to inhibit tumor growth has been demonstrated in many cancers including prostate cancer (PCa). High concentrations of androgens, which inhibit the growth of the LNCaP human PCa cell line, have been shown to have both positive and negative effects on IGFBP-3 expression by different laboratories. To further explore the relationship between IGFBP-3 and androgens, we examined IGFBP-3 expression in LNCaP cells. We demonstrate that IGFBP-3 expression can be induced by 10 nm of the synthetic androgen R1881 or dihydrotestosterone. Transactivation assays show that the 6-kb IGFBP-3 promoter sequence directly responds to androgen treatment. In silico analysis identified a putative androgen response element (ARE) at -2,879/-2,865 in the IGFBP-3 promoter. A single point mutation in this ARE disrupted transactivation by R1881. Combining the data obtained from EMSA, chromatin immunoprecipitation and mutational analysis, we conclude that a novel functional ARE is present in the IGFBP-3 promoter that directly mediates androgen induction of IGFBP-3 expression. Furthermore, we found that the combination of androgens and calcitriol significantly potentiated the IGFBP-3 promoter activity, suggesting that enhanced induction of the expression of the endogenous IGFBP-3 gene may contribute to the greater inhibition of LNCaP cell growth by combined calcitriol and androgens. Because androgens are well known to stimulate PCa growth and androgen deprivation therapy causes PCa to regress, the stimulation by androgens of this antiproliferative and proapoptotic protein is paradoxical and raises interesting questions about the role of androgen-stimulated IGFBP-3 in PCa.

Recombinant, Nonglycosylated Human Insulin-Like Growth Factor-Binding Protein-3 (IGFBP-3) Is Degraded Preferentially after Administration to Type II Diabetics, Resulting in Increased Endogenous Glycosylated IGFBP-3

Administration of IGF-binding protein-3 (IGFBP-3) with IGF-I stabilizes IGF-I concentrations and prolongs its half-life. One determinant of IGFBP-3 stability is proteolysis. Normal subjects have minimal IGFBP-3 protease activity; however, with pregnancy, acute catabolic illness, or diabetes, IGFBP-3 protease activity is increased. This study was conducted to determine the degree of proteolysis that occurs in glycosylated, endogenous serum IGFBP-3 and nonglycosylated IGFBP-3 after administration of an IGF-I/IGFBP-3 combination to patients with diabetes. Thirty-two patients received either 1 (n = 8) or 2 (n = 24) mg/kg x d IGF-I/IGFBP-3 by bolus s.c. injection (n = 16) or continuous s.c. infusion (n = 16). When nonglycosylated IGFBP-3 was given, the abundance of both glycosylated and nonglycosylated forms of IGFBP-3 in serum was increased. Incubation of nonglycosylated IGFBP-3 with diabetic serum in vitro resulted in more rapid degradation compared with glycosylated IGFBP-3. When the serum obtained from subjects who had received nonglycosylated IGFBP-3 was analyzed, significant differences in the stability of glycosylated and nonglycosylated IGFBP-3 were present. The addition of increasing concentrations of nonglycosylated IGFBP-3 to diabetic serum resulted in a dose-dependent increase in the abundance of endogenous, glycosylated IGFBP-3. Administration of IGF-I and nonglycosylated IGFBP-3 for 2 wk to 32 subjects increased glycosylated IGF-I/IGFBP-3 by 20-40%. The increases were the greatest in the groups that received IGFBP-3 by infusion (e.g. 31% and 40%). After administration to diabetics, nonglycosylated IGFBP-3 is degraded more rapidly than glycosylated IGFBP-3. By acting as a preferential substrate for the IGFBP-3 protease, nonglycosylated IGFBP-3 protects endogenous, glycosylated IGFBP-3 from degradation, allowing total IGFBP-3 concentrations to increase.

Characterization of a Novel Positive Transcription Regulatory Element That Differentially Regulates the Insulin-like Growth Factor Binding Protein-3 (IGFBP-3) Gene in Senescent Cells

Insulin-like growth factor binding protein-3 (IGFBP-3) is a well documented growth inhibitor and pro-apoptotic factor. IGFBP-3 mRNA and its protein are overexpressed by senescent human diploid fibroblasts. However, the mechanism responsible for the up-regulation of its expression is still unclear. This report describes a novel transcriptional regulatory element, IGFBP-3 enhancer element (IEE), identified in the 5' untranslated region of the IGFBP-3 gene. This element differentially activates IGFBP-3 expression in senescent versus young fibroblasts. Electrophoretic mobility shift assays revealed abundant complexes in senescent cell nuclear extracts compared with young cell nuclear extracts. Similar to young proliferative cells, young quiescent cells showed reduced binding activity; enhancement of this activity was specific to senescent cells and not an effect of cell cycle arrest. The DNase I footprint revealed the protein-binding core sequence within the IEE through which the protein binds the IEE. Site-directed mutagenesis within IEE abolished binding activity and selectively decreased IGFBP-3 promoter activity in senescent (but not young) cells. Furthermore, introduction of an IEE decoy suppressed the endogenous IGFBP-3 gene expression specifically in senescent cells. These results point to the IEE as being a positive transcription regulatory element that contributes to the up-regulation of IGFBP-3 during cellular senescence.

IGFBP-3 activates TGF-beta receptors and directly inhibits growth in human intestinal smooth muscle cells.

We have shown that human intestinal smooth muscle cells produce IGF-I and IGF binding protein-3 (IGFBP-3). Endogenous IGF-I acts in autocrine fashion to stimulate growth of these cells. IGFBP-3 inhibits the binding of IGF-I to its receptor and thereby inhibits IGF-I-stimulated growth. In several carcinoma cell lines and some normal cells, IGFBP-3 regulates growth independently of IGF-I. Two mechanisms for this effect have been identified: IGFBP-3 can directly activate transforming growth factor-beta (TGF-beta) receptors or it can undergo direct nuclear translocation. The aim of the present study was to determine whether IGFBP-3 acts independently of IGF-I and to characterize the mechanisms mediating this effect in human intestinal smooth muscle cells. The direct effects of IGFBP-3 were determined in the presence of an IGF-I receptor antagonist to eliminate its IGF-I-dependent effects. Affinity labeling of TGF-beta receptors (TGF-betaRI, TGF-betaRII, and TGF-betaRV) with 125I-labeled TGF-beta1 showed that IGFBP-3 displaced binding to TGF-betaRII and TGF-betaRV in a concentration-dependent fashion. IGFBP-3 stimulated TGF-betaRII-dependent serine phosphorylation (activation) of both TGF-betaRI and of its primary substrate, Smad2(Ser465/467). IGFBP-3 also caused IGF-I-independent inhibition of basal [3H]thymidine incorporation. The effects of IGFBP-3 on Smad2 phosphorylation and on smooth muscle cell proliferation were independent of TGF-beta1 and were abolished by transfection of Smad2 siRNA. Immunoneutralization of IGFBP-3 increased basal [3H]thymidine incorporation, implying that endogenous IGFBP-3 inhibits proliferation. We conclude that endogenous IGFBP-3 directly inhibits proliferation of human intestinal smooth muscle cells by activation of TGF-betaRI and Smad2, an effect that is independent of its effect on IGF-I-stimulated growth.

Insulin-like growth factor binding protein-3 is a novel mediator of apoptosis in insulin-secreting cells.

Insulin-like growth factor binding protein-3 (IGFBP-3) is emerging as a critical regulator of cell survival. There has been no study which directly examined the potential role for this major growth factor in the programmed cell death (apoptosis) of insulin-secreting cells. To determine whether IGFBP-3 mediates apoptosis in insulin-secreting cells, we performed a rigorous series of experiments with the rat insulinoma (RIN) cell line m5F and the hamster insulin-secreting tumor (HIT) T-15. Within 24 h exogenous IGFBP-3 induced significant DNA fragmentation in RIN and HIT cells, at doses ranging from 4.4 to 2000 ng/ml (P<0.05) without a classic dose-response relationship. DNA fragmentation induced by rhIGFBP-3 occurred in the presence of immunoglobulin to block the type 1 IGF receptor. As detected by flow cytometry for Annexin V exposure to the cell surface, rhIGFBP-3 treatment doubled the proportion of apoptotic HIT cells from 1.7 +/- 0.4% (serum-free control) to 3.4 +/- 0.2% (P<0.02), an effect completely reversed by co-treatment with 1000 ng/ml rhIGF-I. Immunofluorescent microscopy disclosed that pro-inflammatory Th1 cytokines increased intranuclear aggregation of endogenous IGFBP-3. Cytokine-induced DNA fragmentation was completely blocked by relatively brief pre-treatment with antisense IGFBP-3 phosphorothioate oligodeoxynucleotides. In conclusion, we have presented the first evidence that IGFBP-3 contributes to cytokine-mediated apoptosis in insulin-secreting cells.

Cellular Internalization of Insulin-like Growth Factor Binding Protein-3: DISTINCT ENDOCYTIC PATHWAYS FACILITATE RE-UPTAKE AND NUCLEAR LOCALIZATION

Insulin-like growth factor binding protein-3 (IGFBP-3) is well established as a growth-inhibitory, apoptosis-inducing secreted molecule that acts via insulin-like growth factor (IGF)-independent as well as IGF-dependent pathways. Nuclear localization of IGFBP-3 has been observed and nuclear binding partners for IGFBP-3 demonstrated. However, little is known about the mechanism of IGFBP-3 internalization. We hypothesized that IGFBP-3 is first secreted then taken up again into cells and that its internalization could occur via binding to transferrin or caveolin. Incubation of cells with an IGFBP-3-neutralizing antibody demonstrated that nuclear translocation of endogenous IGFBP-3 requires IGFBP-3 secretion and re-uptake. Nuclear localization of exogenously added IGFBP-3 was rapid, occurring within 15 min, inhibited by co-incubation and extracellular sequestration with IGF-I, and dependent on the transferrin-binding C-terminal peptide region of IGFBP-3. Co-immunoprecipitation assays confirmed that IGFBP-3 binds transferrin but not directly to the transferrin receptor (TfR1); however, transferrin binds TfR1 and a ternary complex is formed. Specific binding to caveolin scaffolding docking sequence was confirmed utilizing radiolabeled IGFBP-3. Blocking TfR1-mediated endocytosis prevents both endogenous and exogenous IGFBP-3 re-uptake and inhibitors of caveolae formation also retard IGFBP-3 nuclear entry. Co-treatment with anti-transferrin receptor antibody and cholesterol depletion agents completely abolished endogenous and exogenous IGFBP-3 uptake. Suppression of IGFBP-3 internalization by TfR1 blockade inhibited IGFBP-3-induced apoptosis. Together, these data indicate that the actions of IGFBP-3 are mediated by internalization via distinct endocytic pathways.

Constitutive expression of IGF-binding protein-3 by mammary epithelial cells alters signaling through Akt and p70S6 kinase.

IGF-binding protein-3 (IGFBP-3) potentiates IGF-I action in the non-transformed mammary epithelial cell line, MAC-T, via a mechanism that is independent of its ability to bind IGF-I. The goal of the present study was to determine if IGFBP-3 might enhance IGF action by influencing intracellular signaling events downstream of the IGF receptor. IGF-I stimulated a time-dependent activation of Akt in which phosphorylation of Ser(473) was detectable by 1 min and maximal at 15 min. In contrast, no activation of extracellular signal-regulated kinase (ERK)1/2 by IGF-I was observed although basal phosphorylation was readily detectable. In MAC-T cells constitutively expressing IGFBP-3 (+BP3), phosphorylation of Akt following stimulation with IGF-I was enhanced relative to mock-transfected cells (Mock). The enhancement was detectable within 1 min of IGF-I treatment and persisted for up to 10 h. The increased phosphorylation observed by Western blotting corresponded to a 1.7-fold increase in Akt kinase activity. The enhanced Akt response was elicited by factors that activate the IGF receptor but exhibit reduced affinity for IGFBP-3, such as Long R(3)IGF-I, B chain IGF-I and insulin. In contrast, [Leu(60)]IGF-I, which binds IGFBP-3 but has reduced affinity for the IGF receptor, failed to induce comparable activation, suggesting that an association between IGF-I and IGFBP-3 is not required for the effect. The enhanced Akt activation could not be mimicked by addition of exogenous IGFBP-3. Akt phosphorylation was also enhanced by transforming growth factor-alpha in +BP3 cells, indicating that the effect was not specific to IGF-I. Similar to Akt, phosphorylation of p70S6 kinase (p70(S6K)) by IGF-I was also enhanced in +BP3 cells relative to Mock cells at both 15 min and 10 h. However, this was largely an effect of lower basal activation of p70(S6K) in +BP3 cells. These data indicate that endogenous IGFBP-3 potentiates IGF action in MAC-T cells by enhancing signaling via the phosphatidylinositol 3-kinase pathway at a point that is downstream of IGF receptor activation. Further studies will delineate specific mechanisms by which IGFBP-3 may influence intracellular events that regulate growth in mammary epithelial cells.

Insulin-like growth factor binding protein-3 (IGFBP-3) potentiates paclitaxel-induced apoptosis in human breast cancer cells

Variability in response to chemotherapy is poorly understood. Paclitaxel-induced apoptosis was assessed in human Hs578T breast cancer cells, using the MTT assay, cell counting, morphological features and flow cytometry. Pre-dosing cells with non-glycosylated insulin-like growth factor binding protein-3 (ngIGFBP-3) had no effect on the cells per se but accentuated paclitaxel-induced apoptosis. The apoptotic pathway was further examined by measuring caspase-3 activity in cell lysates at time points over 48 hr after dosing with paclitaxel. Activity increased significantly, and Western immunoblots for caspase-3 in conditioned media showed that the inactive precursor decreased after incubation with paclitaxel. Endogenous production of IGFBP-3 by the cells after incubation with paclitaxel was evaluated using Western ligand blotting, specific IGFBP-3 immunoblotting and radioimmunoassay. Paclitaxel increased endogenous IGFBP-3, which was further increased if the cells had been pre-dosed with ngIGFBP-3. These findings suggest that IGFBP-3 may be an important modulator of paclitaxel-induced apoptosis.